Molecular Phylogenetics and Evolution 43 (2007) 714–725
www.elsevier.com/locate/ympev
Parallel diversification of Australian gall-thrips on Acacia
M.J. McLeish
a
a,*
, B.J. Crespi b, T.W. Chapman c, M.P. Schwarz
d
South African National Biodiversity Institute, Kirstenbosch Research Centre, Private Bag X7, Claremont, Cape Town 7735, South Africa
b
Behavioural Ecology Research Group, Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada V5A 1S6
c
Memorial University, St. John’s, Newfoundland, Canada A1B 3X9
d
Flinders University, Sturt Road, Bedford Park, Adelaide, South Australia 5042, Australia
Received 3 January 2006; revised 12 March 2007; accepted 14 March 2007
Available online 27 March 2007
Abstract
The diversification of gall-inducing Australian Kladothrips (Insecta: Thysanoptera) on Acacia has produced a pair of sister-clades,
each of which includes a suite of lineages that utilize virtually the same set of 15 closely related host plant species. This pattern of parallel
insect-host plant radiation may be driven by cospeciation, host-shifting to the same set of host plants, or some combination of these
processes. We used molecular-phylogenetic data on the two gall-thrips clades to analyze the degree of concordance between their phylogenies, which is indicative of parallel divergence. Analyses of phylogenetic concordance indicate statistically-significant similarity
between the two clades. Their topologies also fit with a hypothesis of some degree of host–plant tracking. Based on phylogenetic and
taxonomic information regarding the phylogeny of the Acacia host plants in each clade, one or more species has apparently shifted
to more-divergent Acacia host–plant species, and in each case these shifts have resulted in notable divergence in aspects of the phenotype
including morphology, life history and behaviour. Our analyses indicate that gall-thrips on Australian Acacia have undergone parallel
diversification as a result of some combination of cospeciation, highly restricted host–plant shifting, or both processes, but that the
evolution of novel phenotypic diversity in this group is a function of relatively few shifts to divergent host plants. This combination
of ecologically restricted and divergent radiation may represent a microcosm for the macroevolution of host plant relationships and
phenotypic diversity among other phytophagous insects.
! 2007 Elsevier Inc. All rights reserved.
Keywords: Parallel divergence; Cospeciation; Host-switch; Phylogenetics; Gall-thrips; Acacia
1. Introduction
Evolutionary conservation of associations between plant
and phytophagous insect groups is a central theme in biology and provides a platform for testing hypotheses rich in
scope (Futuyma and Moreno, 1988; Jermy, 1993; Kelley
et al., 2000; Craig et al., 2001; Johnson et al., 2002; Nyman,
2002; Ward et al., 2003; Zerega et al., 2005; Jousselin et al.,
2006; McLeish et al., 2007). Coevolution theory (Ehrlich
and Raven, 1964) was the historic impetus driving work
endeavouring to penetrate factors explaining radiations
*
Corresponding author. Fax: +27 0 21 797 6903.
E-mail address: mcleish@sanbi.org (M.J. McLeish).
1055-7903/$ - see front matter ! 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2007.03.007
of both phytophagous insects and their host–plants via
selective responses to one another over a relatively long
period. ‘Coevolution’ has also been used to demonstrate
joint speciation of interacting lineages, or cospeciation
(Herre et al., 1996; Clayton et al., 1999; Page, 2003). However, the extent to which phytophagous insects and the
plants with which they interact exert selection on one
another is complex, highly varied among lineages, and
unclear (Jermy, 1984, 1993; Ballabeni et al., 2003). In this
study, we infer a phylogeny of gall-inducing thrips on Australian Acacia and test hypotheses concerning how this
plant–insect assemblage has evolved.
Gall-inducing insects are tightly constrained to mechanisms by which speciation might proceed. Australian gallinducing thrips are phytophagous insects that have evolved
M.J. McLeish et al. / Molecular Phylogenetics and Evolution 43 (2007) 714–725
strategies permitting specific utilisation of desert Acacia
species for food and shelter and for these reasons gallinduction imposes a level of phylogenetic constraint (Cornell, 1983; Jermy, 1993; Farrell and Mitter, 1998a; Craig
et al., 2001; Ward et al., 2003). Host race formation is
apparent in gall-thrips on Acacia (Crespi et al., 2004). As
well as preadaptation to closely related host plants, cospeciation and host switching across related plants has been
shown to result in life history shifts, host specialisation,
and the macroevolutionary conservatism in resource use
(Ehrlich and Raven, 1964; Berlocher, 2002; Crespi et al.,
1998, 2004; Després and Jaeger, 1999; Cronin and Abrahamson, 2001; Drès and Mallet, 2002; Machado et al.,
2005; Rønsted et al., 2005).
Gall-inducing thrips are a monophyletic group that
inhabit species of Plurinerves, Juliflorae, and Phyllodinae
Acacia subgenera, or sections. Putative gall-thrips species
on closely related hosts are of particular interest. Presumably, these taxa have recently diverged and are expected
to include taxa near or below species-level and provide a
more transparent interpretation of cladogenesis in gallthrips with fewer extinction events obscuring thrips-Acacia
associations. Kladothrips rugosus Froggatt and Kladothrips
waterhousei Mound and Crespi induce galls on the same 14
Plurinerves host species showing a high degree of distribution overlap. The phylogenetic relationships among these
cryptic taxa are yet to be resolved. Cryptic species are different species that cannot be easily distinguished on the
basis of morphology and is indicative of recently diverged
species (Jaenike, 1981; Parsons and Shaw, 2001). The
apparent cryptic species K. rugosus and K. waterhousei
complexes appear overwhelmingly host-specific, they
induce disparate taxon-specific gall morphologies, and preliminary molecular work using COI sequence data and
microsatellites have provided strong evidence for specieslevel divergence among them (Mound, 1971; Mound
et al., 1996; Crespi et al., 1997, 1998; McLeish et al.,
2006). However, some of these putative species show little
genetic divergence, which is suggestive of host race population’s status.
The scope of this work does not include discussion of
species definitions, but contends that levels of polymorphism, below that of species, exist in our dataset and
require elaboration. Genetic distances among K. rugosus
and among K. waterhousei populations show that a large
majority of the K. rugosus and K. waterhousei complex
members are apparently different species, though additional diagnoses would be useful. Measures of gene flow
have to be determined to show reproductive isolation. In
addition to genetic distance, it is also crucial to use behavioural and ecological criteria to identify species (Ferguson,
2002). Both the phylogenetic inferences indicate gall structure is highly conserved amongst all newly sampled populations. It is commonly accepted that gall morphology is
largely under the control of the insect genome and represents an extended phenotype (Stern, 1995; Crespi and Worobey, 1998; Morris et al., 2002; Stone and Schönrogge,
715
2003). Fidelity of gall structure over different host species
is consistent with gall phenotype being largely determined
by the thrips genotype and therefore a potentially useful
diagnostic character in species identification. Species-specificity of gall morphology is evident in other insect orders
(sawflies: Nyman et al., 2000: wasps: Cook et al., 2002).
Recent molecular work (McLeish et al., 2006) has shown
two K. rugosus populations, each of which induces a discrete gall type, once believed to be the same species, are
different.
The K. rugosus and K. waterhousei complexes thus appear
to represent ecological replicates (Johnson and Clayton,
2003) sharing the same set of host species, each expected
to cluster into a separate clade and respond in parallel to
host speciation via cospeciation, host switching and/or host
race formation. These clades thus represent an excellent
opportunity to test for parallel diversification and evaluate
the roles of historical contingency and selection in evolutionary change (Ricklefs and Schluter, 1993).
1.1. Modes of speciation
Speciation in gall-thrips might proceed by the formation
of host-related races where there is reduced gene flow
among populations of a single species parasitising two or
more localised host species leading to reproductive isolation (Jaenike, 1981, 1990; Emelianov et al., 1995; Parsons
and Shaw, 2001; Drès and Mallet, 2002). Speciation via a
host-shift can be thought of as a transition from polymorphism (e.g. for host preference) to host race preceding a
transition from host race to reproductively isolated species.
Host races are maintained by reduced gene flow predominantly via differential host preference. By contrast,
host-related sibling species are reproductively isolated for
reasons in addition to differential host preference (Jaenike,
1981). Genetic divergence data suggests that host-related
races of gall-thrips are actually a series of host specific
sibling species, which is consistent with the strong host–
plant specificity shown in virtually all other gall-inducing
insects (Crespi et al., 2004; Rohfritsch and Shorthouse,
1982).
Cospeciation between phytophagous insects and their
hosts, parasites, or mutualists has been clearly demonstrated in a number of cases, most of which involve strong
host–plant specificity and intimate insect–plant relationships such as gall-induction or complex physiological and
life history adaptation (Ronquist and Nylin, 1990; Baker,
1996; Herre et al., 1996; Machado et al., 1996; Roderick,
1997; Roderick and Metz, 1997; Farrell and Mitter,
1998b; Burckhardt and Basset, 2000; Clark et al., 2000; Itino et al., 2001; Weiblen and Bush, 2002; Weiblen, 2004).
The majority of studies, however, indicate that congruence
between insect and host plant phylogenies is partial or nonexistent, and thus host-shifting appears to be the more prevalent mechanism in determining the associations of insects
and their hosts (Humphries et al., 1986; Weintraub et al.,
1995; Janz and Nylin, 1998; Dobler and Farrell, 1999; Janz
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M.J. McLeish et al. / Molecular Phylogenetics and Evolution 43 (2007) 714–725
et al., 2001; Jones, 2001; Lopez-Vaamonde et al., 2001;
Ronquist and Liljeblad, 2001). Consequent to host-shifting, fitness tradeoffs between hosts, or ecological divergence of derived, host-shifted populations, may spur the
evolution of reproductive isolation (Joshi and Thompson,
1997; Hawthorne and Via, 2001; Nosil et al., 2002), and
colonisation of new host–plant lineages may provide
opportunities to diversify rapidly (Ehrlich and Raven,
1964; Mitter et al., 1988; Farrell and Mitter, 1998b).
Cospeciation between gall-inducing thrips and host Acacia lineages has been suggested at a macroevolutionary
scale in an explicitly phylogenetic context (Crespi et al.,
2004). Two thrips lineages, each producing a morphologically discrete elongate or pouched gall type on Acacia sections Plurinerves and Juliflorae can be traced from two
ancestral gall-inducing species on a single ancestral Acacia
lineage. Both derived thrips lineages have retained the
ancestral elongate-pouched gall type combination. A
hypothesis of cospeciation makes three predictions (Crespi
et al., 2004): (1) phylogenies of parallel thrips lineages
should be identical or very similar to each other, (2) phylogenies of thrips lineages should be identical or very similar to that of the host plants, and (3) speciation events
among parallel thrips lineages and host lineages should
be contemporaneous. Deviations from an identical match
would be indicative of processes other than cospeciation
operating. Alternatively, the K. rugosus and K. waterhousei
groups might have independently converged onto the same
set of closely related Acacia conducive to Kladothrips gallinduction, as host-shifts are reported (Craig et al., 1994) to
more freely occur among taxonomically and phylogenetically similar plants.
Contradicting the model involving complete cospeciation is evidence that indicates the apparent coincidence
in several species of major morphological and life history
changes accompanying host switches between more distantly related host lineages that are not inhabited by closely related thrips sister-species (Crespi et al., 2004). These
switches are also evidenced by the absence of elongatepouched gall type combinations and by the presence of
only a single gall type on the novel host species, as in
Kladothrips intermedius, K. rodwayi and K. morrisi (Crespi
et al., 2004). Convergence of thrips lineages among
related hosts would predict their phylogenies to be independent of one another. In this paper we test for parallel
speciation in the K. rugosus and K. waterhousei species
complexes, and evaluate hypotheses for their joint diversification on Australian Acacia. To do so, we first extend
and revise the current gall-thrips phylogeny (Morris
et al., 2001) with addition of K. rugosus, K. waterhousei,
K. habrus, and K. intermedius ‘races’ from different Acacia
species; and second, use the phylogeny to test for parallel
patterns of diversification between the K. rugsosus and the
K. waterhousei groups, which would be indicative of cospeciation of each of these groups with their Acacia hosts,
or the parallel evolution of the same set of host–plant
shifts.
2. Materials and methods
2.1. Collections, DNA extractions, PCR, and sequencing
Taxa were collected from widely distributed Acacia populations across Australia (Table 1). Voucher specimens of
these taxa have been deposited in the Australian National
Insect Collection (ANIC) at CSIRO Entomology in Canberra. Gall morphology is highly conserved within each
gall-thrips taxon with structural diversity exhibited amongst
them (Crespi and Worobey, 1998), and was used in conjunction with host species identification (Maslin, 2001) to discriminate amongst gall-thrips races. To test whether gall
structure can be used as an indicator of association between
like-types, we mapped three distinct gall structure categories
onto focal taxa in our phylogenies: (1) ‘spiky’ galls have very
obvious pointed protrusions: (2) ‘elongate’ galls are those
that are elongate or tubular, some of which have subtle
surface textural qualities such as fine striations: and
(3) ‘pouched’ galls that form from a ‘ballooning’ of petiole
tissue from one surface of the phyllode with a noticeably
more narrow ostiole than is formed in elongate galls.
Fragments of cytochrome oxidase I (COI), elongation factor — one alpha (EF-1a), wingless, and 16S (ribosomal
RNA) gene regions were sequenced. The DNA extractions
used for the sequencing data were from fresh tissue frozen
to !80". To maximise DNA yield each tissue extraction
comprised of all individuals in a single gall, the brood of
one female (Chapman et al., 2000), using a GENTRA SYSTEMS DNA Extraction Kit. Amplifications of DNA was
undertaken using the following protocol: 94 "C, 45 s denaturation; 48 "C, 1 min annealing; 72 "C, 1 min extension
for 34 cycles; with a final cycle of 72 "C, 6 min extension.
The polymerase enzyme used was Amplitaq Gold (ROCHE)
that required a 90 "C, 9 min incubation period for the first
cycle only. The PCR mixture was a 25 ll reaction including:
1· buffer (ROCHE), 1 U of Amplitaq Gold polymerase. Four
millimeter of MgCl2, 0.8 mM of dNTPs, 5 pmol of each primer, and unknown concentrations of template DNA.
The following primer pairs were used to amplify the various gene fragments. The COI gene fragment was amplified
using two primer pair sets: LCO1490: 50 -GGT CAA CAA
ATC ATA AAG ATA TTG G-30 with HCO2198 50 -TAA
ACT TCA GGG TGA CCA AAA AAT CA-30 (Folmer
et al., 1994) and C1-J-2183 50 -CAA CAT TTA TTT
TGA TTT TTT GG-30 (Simon et al., 1994) with A2735
50 -AAA AAT GTT GAG GGA AAA ATG TTA-30 (Crespi B). The EF-1a gene fragment was amplified using primer
pair sets M51.9 50 -CAR GAC GTA TAC AAA ATC GG30 (Cho et al., 1995) with 50 -AGA CTC AAC ACA CAT
AGG TTT GGA C-30 (Morris D) and G730 50 -ACC
TTC GCT CCT GCC AAC TT-30 with G731 50 -AAG
GGT GAT AAT AGC AGC-30 (McLeish M). The wingless
gene fragment was amplified using primer pair 50 -TAG
ACG TAT CGT TAC ACT GC-30 and 50 -CGT CAA
GAC CTG CTG GAT GC-30 (McLeish M). The 16S (ribosomal RNA) gene fragment was amplified using CI-J-2195
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M.J. McLeish et al. / Molecular Phylogenetics and Evolution 43 (2007) 714–725
Table 1
Description and locations of collection sites for the Kladothrips rugosus and K. waterhousei species complexes used for the sequence data
Galler
Host
Site description
Date
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
K.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
20 km N of Quilpi QLD
15 km S of Yenda NSW
15 km W of Mt. Hopeless SA
25 km W of Whyalla SA
Lk. Bindegolly QLD
20 km E of Dalwallinu WA
3 km W of Merridin WA
25 km NW of Tibooburra NSW
83 km SW Broken Hill NSW
9 km S of Yenda NSW
10 km W of Prairie QLD
40 km E of Quilpie QLD
19 km S of Miles QLD
25 km W of Whyalla SA
95 km S of Griffith NSW
9 km S of Wooramel WA
8 km N of Barcaldine QLD
57 km E of Morven QLD
79 km W of Wilcannia NSW
20 km E of Dalwallinu WA
3 km W of Merridin WA
25 km NW of Tibooburra NSW
45 km N of Adavale QLD
121 km E Quilpie QLD
Lk. Bindegolly QLD
37 km N of Aramac QLD
August 04
May 04
March 04
February 02
April 97
April 97
January 99
April 98
April 05
May 04
April 98
August 04
April 98
February 02
April 04
April 97
February 04
April 97
March 96
April 97
January 99
April 98
April 97
April 98
April 97
March 98
sterni
habrus
intermedius
rugosus
rugosus
rugosus
rugosus
rugosus
rugosus
rugosus
rugosus
rugosus
rugosus
rugosus
rugosus
rugosus
rugosus
waterhousei
waterhousei
waterhousei
waterhousei
waterhousei
waterhousei
waterhousei
waterhousei
waterhousei
aneura
pendula
oswaldii
papyrocarpa
ammophila
ancistrophylla
enervia
cana
loderi
melvillei
microcephala
microsperma
omalophylla
papyrocarpa
pendula
sibilans
tephrina
maranoensis
loderi
ancistrophylla
enervia
cana
microsperma
omalophylla
ammophila
microcephala
For collections details of other species see Morris et al. (2001). Host populations of K. habrus, K. intermedius, and K. waterhousei were also collected.
50 -CCG GTC TGA ACT CAG ATC ACG T-30 (Simon
et al., 1994) with A2735 50 -CGC CTG TTT AAC AAA
AAC AT-30 (Crespi B). We amplified up to 1245 bp of the
COI mitochondrial gene, 444 bp of the EF-1a gene,
472 bp of the 16S, and 549 of the wingless gene. SeqEd
v1.0.3 (http://helix.nih.gov/docs/gcg/seqed.html) was used
to edit sequences. Sequences were aligned using
CustalX 1.81.1a software (Thompson et al., 1997; ftp://
ftp-igbmc.u-strasbg.fr/pub/ClustalX/ accessed 24 June
2005). All nucleic acid sequence data has been lodged
with GenBank under the Accession Nos. AY827474–
AY827481, AY920988–AY921000, AY921058–AY921069,
and DQ246453–DQ246516.
Taxon sampling is known to effect the estimation of substitution parameters. Our data set of 57 taxa was in excess of
the suggested 20, assumed to be appropriate in accounting
for uncertainty caused by too small a sample (Sullivan
et al., 1999). In five instances, the dataset includes replicate
populations of the same ‘host race’ from samples taken at
either different sites or different years. These taxa include
populations of K. sterni, K. waterhousei (on A. papyrocarpa),
K. intermedius, and K. rugosus (on Acacia cana and x2 races
on A. papyrocarpa). We included these populations to verify
expectations of phylogenetic coherence within a taxon.
2.2. Phylogenetic analysis
Phylogenies were inferred to validate the independence
of the K. rugosus and K. waterhousei groups and extend
and revise the current gall-inducing thrips phylogeny. The
current robust well resolved and well-supported gall-thrips
phylogeny (Morris et al., 2001) comprises 21 described species and was generated using maximum parsimony (MP)
and maximum likelihood approaches. Maximum parsimony is used to test the robustness of model-based trees.
We infer MP phylogenies with the addition of 32 new taxa
using the search parameters consistent with Morris et al.
(2001). To accommodate differences in substitution rate
parameters and base compositional bias in our multiple
gene dataset we implemented a model-based maximum
likelihood Bayesian approach. A recent study (Lin and
Danforth, 2004) advocates a Bayesian approach to accommodate substitution and rate dynamics evident in the gallthrips sequence dataset of Morris et al. (2001). We also
conducted Bayesian analyses using a combined dataset
(i.e. no partitioning of the sequence data) in addition to
analyses using separated data. In all cases, nodal support
for poorly and well-supported relationships were invariably
reproduced by the combined Bayesian analyses.
Maximum parsimony and Bayesian inferences were
implemented in PAUP*b4.10 (Swofford, 2002) and MrBayes (MrBayes 3.0b4, Huelsenbeck and Ronquist, 2001),
respectively. Maximum parsimony analysis was implemented using a heuristic search, with TBR (tree bisection-reconstruction), branch swapping on all best trees,
with 100 random sequence additions holding 10 trees held
at each step. We used 500 heuristic search pseudoreplicates
to calculate bootstrap support values (Felsenstein, 1985),
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M.J. McLeish et al. / Molecular Phylogenetics and Evolution 43 (2007) 714–725
using the same search parameters as above for the
pseudoreplicates. Heuristic search starting trees for
branch-swapping were generated using stepwise addition,
swapping on the best trees only.
To accommodate differences in substitution rate parameters and heterogeneity of base composition in our multiple
gene fragment dataset we fitted separate models to different
gene partitions in the Bayesian analysis (Lin and Danforth,
2004). The sequence data used in the MrBayes analysis was
divided into six partitions comprising 1st, 2nd, and 3rd
codon positions of the cytochrome oxidase one (COI) mitochondrial data, with single partitions for each of elongation
factor one alpha (EF-1a), wingless, and the 16S gene fragments. We used a general time reversible (GTR) DNA substitution model with gamma distributed rates with a
proportion of invariant sites. Posterior probabilities and
mean branch lengths are derived from 3000 trees taken
from generations 3.5 to 5.0 million, sampling every 500th
generation. The sampled trees were derived from post burnin generations after the chains had reached apparent stationarity. We ran the Bayesian analysis 3 times to verify the
repeatability of the phylogenetic outcome. We summarised
the repeatability between independent Bayesian runs by
percent variation of the log likelihood arithmetic means
generated for all generations sampled and for the post burnin sample trees. Log likelihood values reached apparent
stationarity rapidly in each of the three Bayesian analyses.
The arithmetic mean of the log likelihood values for all
generations sampled and for post burnin samples were calculated for each Bayesian analysis and the percent variation between them determined. Percent variation between
the arithmetic means between successive Bayesian analyses
was only 0.11% .
The outgroup, Rhopalothripoides (Bagnall) and Dactylothrips (Bagnall), are the most closely related sister-genera
to Kladothrips Froggatt (Morris et al., 2002). The K. rugosus and Kladothrips waterhousei species complexes were
chosen as ingroup taxa as these groups are expected to
have diversified recently and in parallel on the same set
of host Acacia species.
2.3. Codivergence analysis
Conservative associations between two gall-inducing
thrips groups inhabiting the same Acacia host species suggest that the groups have diversified in parallel (Crespi
et al., 2004). A comprehensive host–plant phylogeny is
not available to compare with a gall-thrips phylogeny.
Under a hypothesis of parallel diversification, both groups
are expected to respond to host speciation in tandem. To
address the hypothesis of parallel divergence, congruence
between two gall-thrips phylogenies inhabiting the same
host species was tested. We inferred separate phylogenies
of the K. rugosus, K. acaciae, and K. ellobus group and
the K. waterhousei, K. habrus, and K. hamiltoni group using
a Bayesian approach sampling every 500th of 3 million
generations. Stationarity was reached almost instanta-
neously in our phylogenetic inferences and to reduce computational time, we sampled every 3 million generations
when generating the trees used to test codivergence hypotheses. The data was analysed using a combined approach in
addition to analysing partitioned data using the same priors as described above. Nodal support differences between
the separate and combined approaches were negligible, and
have therefore elected to show inferences generated using
the partitioned analyses. Nearest ancestral sister-species
were used as outgroups and pruned for the cospeciation
analyses. We assumed that both of the inferences were ‘true
phylogenies.’ As codivergence analyses assume that the
phylogenies used represent the true relationships among
taxa, the trees are not collapsed and support values are
given for all bifurcations to demonstrate regions of
uncertainty.
The extent to which host and parasite phylogenies are
congruent can be used to detect ‘coevolution’ or cospeciation. To test how closely the diversification of gall-thrips
has been subject to host speciation we tested for concordance between the phylogenies of: (I) the K. acaciae,
K. ellobus, and the K. rugosus complex; and (ii) the
K. harpophyllae, K. hamiltoni, K. habrus, K. intermedius,
and K. waterhousei complex. Three approaches using computer programs were used, each treating the data different
ways. First, ParaFit (Legendre et al., 2002 http://
www.bio.umontreal.ca/casgrain/en/labo/parafit.html) was
used to test a global null hypothesis that the association
of two trees has been independent. This approach permits
the treatment of the phylogenies ‘symmetrically’ and is
not directed at reconstructing a putative history of the
association. The associations between the phylogenies are
randomised and tested. Phylogenies of the K. rugosus and
K. waterhousei races are transformed into matrices of principle coordinates and then combined with another matrix
describing the associations between the phylogenies. The
significance of a global fit is tested without direct inclusion
of either one of the phylogenies rather focusing on manipulating the associations. To test the global fit between the
K. rugosus and K. waterhousei groups, we implemented
ParaFit using phylogenies of equal length branches; patristic distances generated in PAUP, and likelihood values
from our Bayesian consensus phylogram. By using equal
branch lengths (of 1) we were able to test the fit of the
topologies only. Cospeciation predicts that codivergences
must occur in a contemporaneous manner. The matrices
approach also allows phylogenies to be represented by likelihood or patristic values that account for concordance
subject to branch length variation.
Finally, as the statistical power afforded by matrix-driven approaches, such as ParaFit, was considered less than
optimal as a result of information loss, concordance
between K. rugosus and K. waterhousei phylogenies was
also assessed using randomisation tests implemented in
the event-driven TreeMap 1.0 (http://www.evolve.zoo.ox.ac.
uk/rod/treemap.html; Page, 1994) and TreeFitter (http://
www.ebc.uu.se/systzoo/research/treefitter/treefitter.html;
M.J. McLeish et al. / Molecular Phylogenetics and Evolution 43 (2007) 714–725
Ronquist, 1997) approaches. Tree fitting and tree mapping
methods are ‘asymmetrical’ in their treatment of the two
trees to be tested for congruence. TreeMap (Page, 1994)
and TreeFitter (Ronquist, 1997) are event-based comparisons that enable the maximisation of codivergence events
(equal to 1) explaining the association between the two
phylogenies by down-weighting duplication, sorting, and
switching events (equal to 0 in each case). These events
are of course invalidated when comparing parasite phylogenies that presumably do not have historic host–parasite
associations. The difference between these two approaches
is that TreeMap requires event costs to be treated in a more
inflexible manner where the codivergence cost is strictly less
than that of a duplication. TreeFitter allows a range of cost
events to be explored where both duplication and codivergence can be set to zero. The ability to consider a greater
range of event cost combinations in a single analysis can
be important for understanding the optimal set of events
more likely to represent the mechanisms operating in any
given phylogenetic association. Here, we find both
approaches less than ideal for our ‘parasite–parasite’ association. However, we considered this type of approach a
useful alternative to exploring the data and used each of
the gall-thrips phylogenies as a pseudo-host tree in separate
analyses.
To easily visualise the host associations among the
K. rugosus and K. waterhousei species complex a tanglegram was generated by inferring Bayesian majority rule
consensus phylogenies for each group and connecting taxa
associated with the same host Acacia species. These trees
were used to generate a set of possible codivergence events
as inferred by TreeMap 1.0. TreeMap provides a graphic
utility that generates a tanglegram displaying the associations and putative codivergence events between two phylogenies. The significance of the association between
phylogenies is determined between observed and randomised trees generated from either of the phylogenies being
compared. An approach that randomises a host phylogeny
is intuitive for host-parasite associations but not optimal
when neither tree is a ‘host’ as such. Here, we assume that
either of the phylogenies is likely to closely match that of
the true host phylogeny under cospeciation criteria, where
either of the thrips phylogenies can be used to simulate the
host phylogeny.
3. Results
We extended and revised the phylogeny of Morris et al.
(2001) with the inclusion of 16 putative races (on different
Acacia species) of the K. rugosus complex and 10 putative
races of the K. waterhousei complex that specialise on the
same 14 host Acacia species. Maximum parsimony and
Bayesian inferences are in general agreement and show a
high level of support for each of the clades containing the
K. rugosus and K. waterhousei complexes (Figs. 1 and 2).
Phylogenetic concordance tests between the K. rugosus
and K. waterhousei species complexes showed a significant
719
Fig. 1. A Maximum parsimony phylogeny using COI, EF-1a, wingless,
and 16S genes implementing a heuristic search with tree bisectionreconstruction (TBR) branchswapping, random addition of taxa 100
replicates per search and 10 trees held at each step, and 500 bootstrap
replicates. K. rugosus and K. waterhousei species complexes are highlighted
with grey boxes. Host tree species are abbreviated in brackets as follows:
amm, A. ammophila; anc, A. ancistrophylla; ane, A. aneura; can, A. cana;
ene, A. enervia; lod, A. loderi; mar, A. maranoensis; mel, A. melvillei; mcp,
A. microcephala; msp, A. microsperma; oma, A. omalophyla; ori, A. orites;
pen, A. pendula; pap, A. papyrocarpa; sib, A. sibilans; and tep, A. tephrina.
Taxon codes are as follows: QLD, Queensland and WA, Western
Australia populations; a, population A; b, population B; E, elongate;
P, pouched; and S, spiky gall structures.
level of non-independence. The ParaFit global test of tree
topology only, using equal branch lengths, was significant
(P = 0.01). TreeMap estimates of the maximum number
of observed codivergence events was significantly more
(0.0025 < P(t = 2.93) < 0.001) than 10,000 random tree
associations (t 6 t0.05(1),1, reject Ho of no difference) and
TreeFitter results indicated a maximum frequency of seven
codivergence events between the observed trees was always
significantly more than those of the randomised trees
(where host tree is K. waterhousei group; P = 0.019 and
where host tree is K. rugosus group; P = 0.016).
3.1. Phylogenetic analysis
The addition of 32 new taxa in our phylogenetic inferences yielded results consistent with the general structuring
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M.J. McLeish et al. / Molecular Phylogenetics and Evolution 43 (2007) 714–725
Fig. 2. Bayesian consensus tree analysis of six separately modelled
partitions comprising 1st, 2nd, and 3rd COI codons and separate EF1a, wingless, and 16S sites. Posterior probabilities and mean branch
lengths are derived from 3000 trees taken from a sample of 5 million
generations, sampling every 500th generation. The K. rugosus and
K. waterhousei species complexes are indicated by the grey boxes. Host
tree species are abbreviated in brackets as follows: amm, A. ammophila;
anc, A. ancistrophylla; ane, A. aneura; can, A. cana; ene, A. enervia; lod,
A. loderi; mar, A. maranoensis; mel, A. melvillei; mcp, A. microcephala;
msp, A. microsperma; oma, A. omalophyla; ori, A. orites; pen, A. pendula;
pap, A. papyrocarpa; sib, A. sibilans; and tep, A. tephrina. Taxon codes are
as follows: QLD, Queensland and WA, Western Australia populations; a,
population A; b, population B; E, elongate; P, pouched; and S, spiky gall
structures.
of the most recent published phylogeny (Morris et al.,
2001). Both maximum parsimony and Bayesian inferences
are in general agreement and indicate that the K. rugosus
and K. waterhousei clades form well-supported and well
resolved groups of 100% for each of these nodes in the
Bayesian inference and 99% in the MP inference (Figs. 1
and 2). A majority of the putative K. rugosus and K. waterhousei host races appear to be at various stages of differentiation (Fig. 3). In particular, taxa exhibiting apparent less
than species-level genetic distances with similar gall structures group as clades (unpublished work). Uncorrected
‘‘p’’ distances for COI between K. rugosus population bearing disparate gall structures were in the order of 6–10%
contrasting those among like types with distances not
exceeding 0.8%. Uncorrected ‘‘p’’ distances between the
outgroup and Kladothrips species ranged from 7 to 12%.
Fig. 3. Consensus phylogram using six separately modelled partitions
comprising 1st, 2nd, and 3rd COI codons and separate EF-1a, wingless, and
16S sites. The K. rugosus and K. waterhousei species complexes are indicated
by the grey boxes. Host tree species are abbreviated in brackets as follows:
amm, A. ammophila; anc, A. ancistrophylla; ane, A. aneura; can, A. cana;
ene, A. enervia; lod, A. loderi; mar, A. maranoensis; mel, A. melvillei; mcp,
A. microcephala; msp, A. microsperma; oma, A. omalophyla; ori, A. orites;
pen, A. pendula; pap, A. papyrocarpa; sib, A. sibilans; and tep, A. tephrina.
Taxon codes are as follows: QLD, Queensland and WA, Western Australia
populations; a, population A; b, population B; E, elongate; P, pouched; and
S, spiky gall structures.
Both MP and Bayesian inferences grouped ‘pouched,’
‘elongate,’ and ‘spiky’ gall structures into clades of like
types. Replicate taxon samples of populations with the
same host and gall structure collected from different sites
or seasons grouped as sister-taxa. These grouping confirmed the expectation that such populations, particularly
at relatively recent stages of differentiation, maintained
phylogenetic coherence. For example, although currently
considered host races, populations of K. waterhousei inhabiting A. cana and A. papyrocarpa sampled across different
years and multiple sites, group together.
The K. rugosus complex was paraphyletic with respect to
K. maslini and the K. waterhousei complex was paraphyletic
with regard to the putative host races of K. habrus and
K. intermedius. The maximum parsimony inference places
Kladothrips rodwayi into a paraphyletic relationship with
the K. waterhousei complex. We suspect low bootstrap
M.J. McLeish et al. / Molecular Phylogenetics and Evolution 43 (2007) 714–725
721
Fig. 4. Bayesian Consensus phylogenies of the K. rugosus and K. waterhousei groups plus associated species. Posterior probabilities are indicated on
branches. Host species are indicated under thrips taxon names. Lines connected to two phylogenies show host associations. Circles on nodes show
codivergence events as inferred by TreeMap 1.0.
support and/or long-branch attraction, due to incomplete
sequence data for the K. waterhousei complex at EF-1a
and wingless gene regions (Wiens, 2005), might have contributed to this outcome. The clade comprising K. acaciae,
K. ellobus, and the K. rugosus complex, matches closely
with host affiliation by the sister-clade comprising
K. harpophyllae, K. hamiltoni, and the K. waterhousei complex. Sister-species from each of these clades specialise on
the same host species, Acacia cambadgei (Baker) and
Acacia harpophylla (Muell, Benth) with the K. rugosus
and K. waterhousei complexes sharing the same set of host
species. Monophyly of the K. rugosus complex becomes
invalid by the presence the K. maslini lineage, though a host
shift is strongly suspected along this lineage (Crespi et al.,
2004).
3.2. Codivergence analysis
ParaFit tests a global null hypothesis that the association between the phylogenies has been independent
(P = 0.01). Tests on individual associations indicate that
this inferred cospeciation was partial rather than complete.
Portions of the two trees are apparently independent as 3
of the 12 links were significant in their contribution to
the global test statistic. Global tests using patristic distances and likelihood values were non-significant at the levels of P = 0.08 and P = 0.18, respectively, suggesting that
non-contemporaneous associations invalidated the significant codivergence events or that molecular-evolutionary
rates differ between lineages in the two clades.
Under the assumption that one of the thrips phylogenies
was equal to the true host phylogeny, both thrips phylogenies were used to simulate the host phylogeny in separate
analyses in TreeMap 1.0 and TreeFitter. An exact search
algorithm implemented in TreeMap 1.0 produced 24 solutions explaining the historical relationship between the phylogenies. All required seven codivergence events, when the
K. rugosus group was assumed to represent the true host
phylogeny. One such solution is summarised in Fig. 4 and
it also shows gall-thrips taxa that share the same host species. An exact search generated 19 solutions all incorporating seven codivergence events when the K. waterhousei
group was assumed to represent the true host phylogeny.
Both trees were randomised using a Markovian model to
generate a distribution (n = 10,000) of codivergence events.
The results indicated a significant (t 6 t0.05(1),1, reject Ho of
no difference) association between the trees. This outcome
indicates that cospeciation apparently occurred more often
than by chance. A randomisation approach implemented in
TreeFitter was used to test the significance of codivergences
in the observed tree associations in two separate analyses,
where alternate phylogenies were assumed to match the true
host phylogeny. Tests of congruence against a randomised
set of trees indicated a significant association (where host
tree is K. waterhousei group; P = 0.019 and where host tree
is K. rugosus group; P = 0.016).
4. Discussion
We inferred phylogenies including numerous undescribed putative species of gall-inducing thrips to test
hypotheses of diversification. The addition of these taxa
to a previous gall-thrips phylogeny (Morris et al., 2001),
results in a tree that includes virtually all known taxonomic
entities for this group. A clear pattern has emerged from
this expanded tree, suggesting that diversity for this group
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M.J. McLeish et al. / Molecular Phylogenetics and Evolution 43 (2007) 714–725
of specialist insects is generated in close association with
host speciation. Maximum parsimony and Bayesian inferences group the K. rugosus and K. waterhousei species complexes as sister-taxa with a high level of support. Significant
congruence between the phylogenies of the K. rugosus and
K. waterhousei groups, including sister-species within each
(Fig. 4), indicated both cospeciation and convergence of
thrips across host–plant species of Acacia in the section
Plurinerves. Codivergence between these thrips groups indicates they were subject to the same isolating events
imposed by host speciation. Poor resolution among
recently diverged taxa within the two complexes renders
cospeciation inferences ambiguous for some associations.
Host switching and the formation of host races among closely related species of this Acacia section does not appear
to be accompanied by the relatively large shifts in adaptive
change when switching between host sections (Crespi et al.,
2004) as implied for K. maslini and K. rodwayi. Highly similar morphologies and marginal adaptive shifts among
thrips belonging to the K. rugosus complex belie substantial
genetic differentiation evident in the COI gene and the
structures of the galls induced by each. This work suggests
that the potential of specialist phytophagous insects to
diversify phenotypically increases with their ability colonise
more distantly related hosts.
4.1. Phylogenetic analyses
Phylogenetic inferences reveal patterns consistent with
some taxa belonging to the K. rugosus and K. waterhousei
complexes being genetically differentiated below the level
evident among the described gall-thrips taxa (Morris
et al., 2001; Crespi et al., 2004; McLeish et al., 2006). These
recently diverged groups offer an opportunity to test codivergence hypotheses where evolutionary processes such as
extinction are unlikely to obscure interpretation, as might
be expected amongst relatively older lineages (Brown
et al., 1995).
The morphological difference between the K. intermedius
samples with ‘pouched’ and ‘elongate’ gall types, was considered negligible (personal communication, Mound LA)
though genetic differentiation of 5% (uncorrected ‘‘p’’ distance for COI) suggests considerable differentiation. The
K. sterni populations from Western Australia (WA) and
Queensland (QLD) grouped as sister-taxa with a high level
of support (Figs. 1and 2) although branch length estimates
(Fig. 4) indicate genetic divergences consistent with allopatric factors. The grouping of K. habrus with putative
K. intermedius populations requires further attention to
establish taxonomic boundaries and nomenclature. Indeed,
lack of phylogenetic signal evident for taxa within these
groups compounds tests of phylogenetic concordance due
to considerable levels of uncertainty (Fig. 4). However, this
ambiguity does not invalidate a signal that was strong
enough to produce a significant level of non-independence
in tests of congruence between these parallel clades affiliated with the same host species.
4.2. Diversification
It has been hypothesized that cospeciation and host
switching processes are responsible for the genetic, phenotypic, and ecological differentiation in gall-thrips (Crespi
et al., 2004). Strong agreement among phylogenetic inferences indicates that the K. rugosus and K. waterhousei complexes are both paraphyletic and inhabit Plurinerves host
species (Figs. 1and 2). These two thrips clades might have
diversified in parallel via each shifting to related, nearby
hosts without cospeciation per se, but broad patterns of
gall-thrips lineages tracking host diversification and evidence of parallel diversification between thrips and host
species at lower scales appears to be partially an outcome
of cospeciation.
The best available Acacia host phylogeny (Crespi et al.,
2004) provides some support for the relationships indicated
by the K. rugosus and K. waterhousei groups and associated
sister-species (Fig. 4). Acacia harpophyllae and A. cambagei
are sister-taxa and are hosts to basal sister-species of both
thrips clades. Although the phylogenetic relationships
among the other Plurinerves species are weakly supported,
this group forms a sister clade to A. harpophyllae and
A. cambagei. Switching between host plants more distantly
related than hosts of gall-thrips sister-taxa, is accompanied
by noticeable life history shifts, is very likely to occur
between hosts that are taxonomically and phylogenetically
close, and have overlapping or adjacent ranges (Maslin,
2001; Crespi et al., 2004). Several examples stand out.
Within the clade also comprising the K. waterhousei group,
losses in sociality (Morris et al., 2001) accompanied by an
apparent switch to more distantly related host have been
inferred (Crespi et al., 2004). Kladothrips xiphius is believed
to represent a loss in sociality and is found on a species
belonging to the Juliflorae section, not Plurinerves as do sister-species. Similarly, K. rodwayi is recognised as a loss in
sociality and it too is found on a more distantly related
host, A. melanoxylon (Fig. 4), a species distributed in temperate and not arid climates. Kladothrips intermedius
inhabits a host species that appears not to be as closely
related to the hosts of thrips sister-species. Like K. rodwayi,
K. intermedius ecloses within the gall, contrasting other
members of this thrips clade that instead disperse as pupa.
Unlike its K. rugosus sister-members on a Plurinerves host
and disperse as pupa, K. maslini inhabits a Juliflorae host
(Figs. 1 and 2) and ecloses as an adult within the gall.
Uncorrected ‘‘p’’ distance (unpublished work) variation
shown by these additional taxa implies both similar and
below those between described gall-thrips species.
The frequency and causes of those host switches traversing host sections accompanying relatively large life history
changes is unknown. A period of host radiation may contribute to gall-thrips ability to switch among host–plants
under speciation (Craig et al., 1994). It appears that the
opportunity for gall-thrips to diversify might have been
assisted by speciation in Plurinerves hosts presumably during a rapid radiation as widespread aridity developed in the
M.J. McLeish et al. / Molecular Phylogenetics and Evolution 43 (2007) 714–725
early Quaternary (Maslin and Hopper, 1982; Clapperton,
1990; Lovejoy and Hannah, 2004). It is expected that
gall-inducing insects should radiate into arid habitats as
pressures exerted by parasitoids, predators, and pathogens
are not as acute in xeric environments (Fernandes et al.,
1994; Blanche and Westoby, 1995; Price et al., 1998; Veldtman and McGeoch, 2003). Radiations into novel niches to
relieve pressures exerted by natural enemies is consistent
with Ehrlich and Raven’s (1964) ‘escape and radiate’
hypothesis predicting the generation of diversity in phytophagous insects as a consequence of strong selection after
colonising a novel host.
4.3. Bimodality in divergence
The tanglegram in Fig. 4 shows phylogenies of both the
K. rugosus and K. waterhousei complexes connected by
lines that join terminal taxa that share the same host species. One of many possible inferred solutions explaining
codivergence events between the trees is indicated at particular bifurcations. Tests for concordance indicate that these
phylogenies are significantly non-independent, but a notable degree of incongruence is also evident. However, phylogenetic uncertainty in the trees might contribute to a
component of ambiguity in the interpretation of codivergence events possibly caused by inclusion of taxa in the
phylogenies that are marginally divergent. Synchronous
genetic isolation between host and parasite can occur at
the level of individual, population, species, or higher
(Rannala and Michalakis, 2003). Codivergence indicates
a frequent incidence of isolating events affecting both thrips
clades in tandem and that cospeciation between gall-thrips
and Acacia operates in a broad sense between lineages and
also between species. A lack of identical fit between the
phylogenies shows independent isolating events has possibly acted on either clade at some stage. Non-significant
contemporaneous branching episodes between the K. rugosus and K. waterhousei groups shown by the ParaFit outcomes for patristic and likelihood branch length values
suggest bimodality in divergence processes. That is, each
thrips clade appears to have responded somewhat differentially to isolating events of the host. Given cospeciation,
one might expect that the more closely related host species
would reflect proportionally similar divergences paralleled
in both thrips lineages that inhabit these hosts, but this pattern does not appear to be consistent with our data.
Bimodality in divergences might reflect differential hostutilisation, dispersal ability, or community driven differences such as escaping natural enemies and interspecific
competition (Jaenike, 1990). For example, demographic
and life history differences between the K. rugosus and
K. waterhousei complexes, such as social organization
(Crespi, 1992) and comparative brood sizes (Wills et al.,
2001) could be invoked to develop hypotheses explaining
this bimodality. There is general agreement between
gall-thrips and best available host phylogenies (Crespi
et al., 2004) but interpretations are also conditional on
723
the presence of a degree of phylogenetic uncertainty. The
ability to test concordance between gall-thrips lineages
has provided insight into mechanisms driving diversification in this group where comparisons of insect and plant
phylogenies were not feasible. This work shows that diversification in phytophagous insects can proceed via a combination of synchronous divergence episodes between insect
and host lineages in addition to independent modes of speciation among them. Speciation by host switching and host
race formation, concurrent with cospeciation, can play a
role in generating diversity. The potential for a group of
specialist phytophages to diversify appears to be closely
linked to their ability to traverse genetic, phenological,
chemical, and morphological obstacles among plant species
varying in susceptibility to colonisation. These patterns
imply that the ability to overcome difficult barriers to colonising novel host plants might determine the degree of
diversity attained in phytophagous insects. Switching to
more distantly related hosts should be accompanied greater
potentials to diversify.
Acknowledgments
We thank the Evolutionary Biology Unit, South Australia Museum, for their sequencing facilities and technical
support. This project was made possible by part funding
from the Nature Foundation SA Inc. (Project #7324), the
Sir Mark Mitchell Research Foundation (CXS10423800),
the Australian Research Council (ARC) to Schwarz
M.P., Cooper J.B., Crespi B.J., Chapman T.W.,
(DP0346322), an ARC Postdoctoral Fellowship to Chapman T.W., and an NSERC grant to Crespi.
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